Ion channels

Ion channels structure & function. Basics of neural science.

1. Ion channels in health and disease Presenter – Dr. Narendiran . S Chair Person – Dr. Saraswati Nashi

2. Introduction Signaling in the brain depends on the ability of nerve cells to respond to very small stimuli . Receptors in the eye respond to a single photon of light . Olfactory neurons detect a single molecule of odorant . Hair cells in the inner ear respond to tiny movements of atomic dimensions. These rapid changes are mediated by ion channels .

3. What are ion channels? Protein molecules that span across the cell membrane Allow passage of ions from one side to the other . An aqueous pore becomes accessible after conformational change.

4. History In the late 1800s, the chemical mechanism underlying nerve and muscle tissue messaging was not known. Ludimar Hermann was able to conclude that nerve and muscle cells were capable of exhibiting a "self- propagating wave of negative charge which advances in steps along the tissue "

5. History In 1880s, Sydney Ringer used a solution of water and ran it through the vessels of an isolated heart from a frog and discovered that in order for the heart to continue beating salts needed to be present in the water. Sodium, calcium, and potassium salts were important and had to be in specific concentration relative to each other .

6. History In 1970s, the existence of ion channels was confirmed by the invention of ‘patch clamp’ technique by Erwin Neher and Bert Sakmann who won a Nobel Prize for it .   In 2003, the Nobel Prize was awarded to American scientists, Roderick MacKinon and Peter Agre for their x-ray crystallographic structure studies on ion channels .

7. Why do we need ion channels? Ions cannot diffuse across the hydrophobic barrier of the lipid bilayer . Provide a polar environment for diffusion of ions across the membrane . Principles of Neural science 5 th edition

8. Specialized functions of ion channels Mediate the generation, conduction and transmission of electrical signals in the nervous system Control the release of neurotransmitters and hormones Initiate muscle contraction Transfer small molecules between cells (gap junctions) Mediate fluid transport in secretory cells Principles of Neural science 5 th edition

9. Questions to be asked? Why do nerve cells have channels? How can channels conduct ions at such high rates and still be selective? How are channels gated? How are the properties of these channels modified by various intrinsic and extrinsic conditions ?

10. Ion channels Vs Ion pumps Pump moves an ion, or a group of a few ions across the membrane. Undergo series of conformational changes. Flow through pumps is 100 to 100,000 times slower. Requires energy to function . Principles of Neural science 5 th edition

11. Ion channels Ion pumps Ion channels Vs Ion pumps

12. Properties Ion channels have three important properties : Specificity & selectivity for specific ions Respond to specific electrical, mechanical, or chemical signals. Conduct ions rapidly across the membrane . Conduct upto 100 million ions per second . Principles of Neural science 5 th edition

13. How is a channel selective?? Based on the size of the pore and ions? Selectivity filter?? Is it carrier based??

14. Armstrong and Hill, Neuron, 1998

15. How is a channel effective?? Cabral et al, Nature, 2001

16. Gating Principles of Neural science 5 th edition

17. Gating Principles of Neural science 5 th edition

18. Gating Principles of Neural science 5 th edition

19. Ligand gated Principles of Neural science 5 th edition

20. Phosphorylation gated Principles of Neural science 5 th edition

21. Voltage gated & Pressure gated Principles of Neural science 5 th edition

22. Functional states Principles of Neural science 5 th edition

23. Energy for gating In voltage-gated channels the energy is provided by the movement of voltage sensor membrane’s electric field. In transmitter-gated channels gating driven by change in chemical-free energy that results when the transmitter binds to channel. For mechanically activated channels the energy associated with membrane stretch is thought to be transferred to the channel. Principles of Neural science 5 th edition

24. Modifiers of gating Principles of Neural science 5 th edition

25. Modifiers of gating Hence modifiers can be Reversible Irreversible Exogenous regulator binding at a different site . Binding at the same site as the endogenous ligand Principles of Neural science 5 th edition

26. Structure of ion channels Hetero- oligomers from distinct subunits. Homo- oligomers from a single type of subunit. A single polypeptide chain organized into repeating motifs Catterall WA, Science, 1988

27. Diversity of ion channels

28. Gene superfamilies Human genome contains Sodium channels - 9 genes Calcium channels - 10 genes Potassium channels - 75 genes Ligand gated channels - 70 genes Chloride channels - 12 genes Members of each gene superfamily have similar Amino acid sequences Transmembrane topology Physiological functions Principles of Neural science 5 th edition

29. Gene superfamilies Principles of Neural science 5 th edition

30. Different Genes Encode Different Pore-Forming Subunits Principles of Neural science 5 th edition

31. Different Pore-Forming Subunits Combine in Various Combinations Principles of Neural science 5 th edition

32. Same pore forming subunit combines with various accesory units

33. Alternative splicing of Pre-mRNA

34. Post-Transcriptional Editing of pre-mRNA

35. Classification Voltage gated – Voltage gated sodium channels. Voltage gated potassium channels. Voltage gated calcium channels. Voltage gated chloride channels . Ligand gated – Nicotinic" Acetylcholine receptor, Ionotropic glutamate-gated receptors and  ATP-gated P2X receptors, and Anion-permeable γ- aminobutyric acid gated GABA A  receptor Other gated channels – Inward-rectifier potassium channels Light-gated channels like channel rhodopsin Mechanosensitive channels Temperature gated channels Cyclic nucleotide gated channels Glial channels. The lancet, 2002

36. Voltage gated Sodium channel Single polypeptide chain . Four homologous domains 6 transmembrane spanning alpha helixes. Whereas potassium channels have 4 separate subunits Payandeh , Nature, 2011

37. Sodium channels Responsible for the rising phase of the action potential. 10 different genes. 4 in the CNS - SCN1A, SCN2A, SCN3A & SCN8A. 3 in PNS – SCN9A, SCN10A & SCN11A . 3 in muscles - SCN4A, SCN5A, & SCN7A. Neuroscience Exploring the brain, 4 th Edition

38. Potassium channels Selective permeability of potassium channels is a key determinant of the resting membrane potential . 4 types of potassium ion channels . Voltage gated potassium channels Calcium-activated K+ channels ( KCa ) Inwardly rectifying K+ channel subunits ( Kir ) 2 pore forming potassium channels (K2P) Bradley’s neurology in clinical practice 7 th edition

39. Potassium channels Neuroscience Exploring the brain, 4 th Edition

40. VGKC - Subtypes KCNA (Shaker), shal , KCNB ( shab ), KCNC ( shaw ), KCND, KCNE, KCNF, KCNG, KCNQ, KCNS, & HCN. Disease causing in humans – KCNA & KCNQ.

41. VGKC complex Nature Reviews , Neurology  2017

42. CASPR2 Complex Principles of Neural science 5 th edition

43. CASPR2 Complex Nature Reviews , Neurology  2017

44. Calcium channels Essential in neuronal signaling & synaptic transmission. 10 different genes. Family 1 - Ca 1.1 to Ca 1.4 mediate the L-type voltage-dependent calcium. Family 2 - Ca 2.1, Ca 2.2 & Ca 2.3 mediates neurotransmission at fast synapses. Family 3 – Ca 3.1, Ca 3.2 & Ca 3.3 Bradley’s neurology in clinical practice 7 th edition

45. Calcium channels - Nomenclature Bradley’s neurology in clinical practice 7 th edition

46. Voltage gated chloride channels Dutzler , FEBS Lett, 2004

47. Ligand - gated Channels activated by neurotransmitter-binding Pentameric AchR GABA Glycine Serotonin Tetrameric NMDA AMPA Kainate Trimeric ATP gated P2X receptors Lemoine et al, Chemical Reviews , 2012 

48. Ligand gated ion channels Chemical Reviews  2012  112  (12), 6285-6318

49. Acetylcholine Receptor Miyazawa et al, Nature,2003

50. Acetylcholine Receptor Miyazawa et al, Nature,2003

51. Glutamate receptors Principles of Neural science 5 th edition

52. Gap junctions Maeda, Nature, 2009

53. Mechanoceptors Channels can be directly activated by forces. Forces conveyed through structural proteins. Forces conveyed to a force sensor & then via second messenger. Principles of Neural science 5 th edition

54. TRP channel Osmolarity pH Temperature

55. TRP channel Bautista DM et al, 2007. Nature

56. Functions of Ion channels

57. Ion channels at each step Principles of Neural science 5 th edition

58. Proc. Natl. Acad. Sci. 1999

59. Synaptic transmission Chemical synapses – Neuromuscular junction Electrical synapses – Gap junctions Principles of Neural science 5 th edition

60. Chemical synapse Neuroscience Exploring the brain, 4 th Edition

61. Neuromuscular junction Principles of Neural science 5 th edition

62. Neuromuscular junction Principles of Neural science 5 th edition

63. EPSP Neuroscience Exploring the brain, 4 th Edition

64. IPSP Neuroscience Exploring the brain, 4 th Edition

65. Functions of Gap junctions Transmission across electrical synapses is extremely rapid . Speed is important for escape responses . Useful for orchestrating the actions of large groups of neurons . Tail flip response of a gold fish. Inking response of Aplysia (snail) Generation of saccades Gap junctions are important for myelination Enhance communication between schwann cell Support layers of myelin Promote the passage of nutrients, metabolites and ions. Principles of Neural science 5 th edition

66. Potassium spatial buffering Extracellular potassium concentrations rise, during periods of neural activity. Increasing extracellular potassium depolarizes neurons. Astrocytes fill most of the space between neurons in the brain. They have gap junctions and inwardly rectifying ( kir ) potassium channels. Excess extracellular potassium taken inside and dissipated over large area of the astrocytes . This is called potassium spatial buffering. Neuroscience Exploring the brain, 4 th Edition

67. Potassium spatial buffering Devinsky , Orrin et al. Trends in Neurosciences

68. Inner ear Kawashima Y et al, 2011. J Clin Invest

69. Inner ear Kawashima Y et al, 2011. J Clin Invest

70. Inner ear Lumpkin EA et al, 1998. J Neurosci

71. LGIC & pain Chemical Reviews  2012 

72. Ion channels and disease

73. There are several basic principles that apply to disorder of channel function. Location selectivity Channel interdependency Genetic heterogeneity Phenotype heterogeneity. Gain of function Loss of function Dominant negative effect Ion channels and disease Celesia et al, clin neurophysio , 2001

74. Ion channels and disease Various mutations within a single gene can lead to distinct clinical syndromes. Mutations in different genes may result in a single recognized clinical entity. Bradley’s neurology in clinical practice 7 th edition

75. Epilepsy & Ion channels Oyrer et al, Pharmacol Rev, 2018

76. Spectrum of channelopathies Bradley’s neurology in clinical practice 7 th edition

77. Inherited channelopathies - Muscle Bradley’s neurology in clinical practice 7 th edition

78. Inherited channelopathies - Neuronal Bradley’s neurology in clinical practice 7 th edition

79. Acquired channelopathies Bradley’s neurology in clinical practice 7 th edition

80. Pain and Channelopathy Bennett, David L H et al. The Lancet Neurology

81. Inherited potassium channelpathies

82. Potassium channel and disease Proc. Natl. Acad. Sci. USA 96 (1999)

83. Episodic Ataxia - 1 Autosomal dominant disease. Characterized by ataxic gait & jerking extremity movements that last for seconds to minutes. Also called episodic ataxia with myokymia Provoking factors - motion & exercise. Episodic failure of excitation of cerebellar neurons & sustained hyperexcitability of the peripheral motoneurons . Treatment - Carbamazepine Bradley’s neurology in clinical practice 7 th edition

84. Mutations in EA - 1 Bradley’s neurology in clinical practice 7 th edition

85. Benign familial neonatal convulsions Incidence is about 1 in 100,000 . Seizures begin a few days after birth. Most patients remit by the age of 4 months 16% of patients continue to suffer seizures in adult life. Mutations in KCNQ2 & KCNQ3. carbamazepine and phenytoin are generally effective in treating BFNC. Bradley’s neurology in clinical practice 7 th edition

86. Mutations in BFNC Bradley’s neurology in clinical practice 7 th edition

87. Isaac’s syndrome Morvan’s syndrome VGKC complex encephalitis Acquired potassium channelopathies

88. Isaac’s syndrome Painful muscle cramps, myotonia & hyperhidrosis . EMG – Myokymic and neuromyotonic discharges. Associated with thymoma . Antibodies against VGKC. Morvan’s syndrome – Neuromyotonia with fluctuating delirium.

89. VGKC encephalitis CASPR2 Encephalitis LGI1 encephalitis Nature Reviews , Neurology  2017

90. VGKC as catecholamine excess

91. Sodium channel and disease Neuroscience Exploring the brain, 4 th Edition

92. GEFS+ Autosomal dominant febrile seizures . Febrile seizures extending beyond 6 years of age. Other seizure types – absence, atonic , myoclonic & partial. EEG - 2.5- to 4-Hz generalized spike-and-wave or polyspike -wave discharges. SCN1B, SCN1A, SCN2A, SCN9A. Bradley’s neurology in clinical practice 7 th edition

93. Mutations in GEFS+ Bradley’s neurology in clinical practice 7 th edition

94. Hyperkalemic periodic paralysis Episodic weakness precipitated by hyperkalemia . Weakness is milder than hypoKPP . Respiratory & ocular muscles remain unaffected. Frequency – several per day to several per year. Attacks are brief – 15 – 60 mins . Triggers – Rest after exercise, food with high K, stress & fatigue. Myotonia + Mutations in SCN4A. Cannon, SC Neuron, 1991

95. Mutations in HyperKPP Bradley’s neurology in clinical practice 7 th edition

96. Paramyotonia congenita Paradoxical myotonia , cold-induced myotonia , & weakness after prolonged cold exposure. Warm up phenomenon negative. Facial muscles, hand & neck muscles commonly affected. Triggers – cold, stress, rest after exercise. Mutations in SCN4A gene . Bradley’s neurology in clinical practice 7 th edition

97. Primary Erythromelalgia Rare autosomal dominant neuropathy. Recurrent burning pain, warmth and redness of the extremities. Two missense  SCN9A  mutations were recently identified. Both mutations cause a hyperpolarizing shift in the voltage-dependence of channel activation and slow the rate of deactivation Triggers - Exertion, heating of the affected extremities, alcohol or caffeine consumption, and any pressure applied to the limbs. Fertelmann CR et al, 2006. Neuron

98. Inherited calcium channelopathies

99. Calcium channelopathy Bradley’s neurology in clinical practice 7 th edition

100. Episodic ataxia type 2 EA2 also called episodic ataxia without myokymia . Age of onset - late childhood or adolescence. Triggers - Physical or emotional stress . Intermittent attacks of vertigo, headache. Interictal nystagmus & impaired vestibulo -ocular reflex . Attacks may be prolonged, lasting for hours and sometimes days. Neuroimaging - cerebellar atrophy. Treatment - Acetazolamide therapy . Newsome-Davis et al, 2003. Ann NY Acad Sci

101. Familial Hemiplegic Migraine Autosomal dominant. Migraine characterized by lateralized motor weakness. 3 classes – FHM type 1, 2 & 3. FHM 1 – Severe type. Auras always involve additional symptoms including sensory, visual, & language disturbances. Aura may last for days with fever, meningismus and cerebellar signs. FHM 2 & FHM 3 – No cerebellar signs. Less severe. Newsome-Davis et al, 2003. Ann NY Acad Sci

102. Spino cerebellar ataxia - 6 Autosomal dominant cerebellar ataxia. CAG trinucleotide repeat in the gene coding for the alpha1A-subunit of the voltage dependent calcium channel. Mean of age at onset – 50 . Prominent cerebellar signs. Intermittent diplopia . Eye signs – Dysmetria , impaired smooth pursuits, impaired ocular movements, peripheral neuropathy. Newsome-Davis et al, 2003. Ann NY Acad Sci

103. Acquired calcium channelopathies

104. Lambert – Eaton Myasthenic syndrome Produces weakness by obstructing neuromuscular transmission. Pathology – Presynaptic . Autoantibodies against P/Q type VGCC. Autonomic dyfunction + Autoantibodies don’t cross the BBB. Paraneoplastic to SCLC. Bradley’s neurology in clinical practice 7 th edition

105. Paraneoplastic cerebellar degeneration Rapid progressive ataxic syndrome. Commonly occurs in cases of breast, ovarian, and lung malignancies. Purkinje cell loss is the pathological hallmark . Anti-P/Q-type VGCC antibodies. May or maynot have neuromuscular involvement. No episodic symptoms. Bradley’s neurology in clinical practice 7 th edition

106. Chloride channelopathies Autosomal dominant – Thomsen’s disease Autosomal recessive – Becker’s disease. Main feature is myotonia - delayed muscle relaxation after contraction. Pronounced after a period of rest, prominent in the legs. Warm up phenomenon positive. Recessive – More common, develop progressive myopathy, severe disease. Pathophysiology Mutation in CLCN1 gene coding for chloride channel. Diminished sarcolemmal chloride conductance. Bradley’s neurology in clinical practice 7 th edition

107. Comparison of channelopathies Bradley’s neurology in clinical practice 7 th edition

108. Mediate intercellular communication Converts the binding of a neurotransmitter released from the presynaptic terminal into an ion flux in the postsynaptic membrane . Have orthosteric -binding site for the NT & an ion channel. Well-established role in neurotransmission. Conventional neurotransmitters - glutamate, acetylcholine, glycine , ATP, serotonin & GABA. Ligand gated Ion channels

109. LGICs and disease

110. Alzheimer’s disease Neurodegenerative disorder characterized by progressive cognitive decline. Loss of neurons & cholinergic synapses in the basal forebrain, cerebral cortex, & hippocampus . Extracellular accumulation of senile plaques. Formation of neurofibrillary tangles composed of hyperphosphorylated tau protein. Aβ neurotoxicity is due in part to complex interactions with nAChRs .

111. Alzheimer’s disease Chemical Reviews  2012 

112. ADNFLE Clusters of brief partial seizures that occur during light sleep. Hyperkinetic tonic stiffening and clonic jerking movements. Aura may precede seizures - somatosensory , sensory, & psychic phenomena . ADNFLE is often mistaken for benign nocturnal parasomnia or night terror. Nocturnal video polysomnography is very useful . Point mutation in the nAChR α4- subunit Chemical Reviews  2012 

113. Congenital myasthenic syndromes Engel AG, Curr Opin Pharmacol 2005

114. Congenital myasthenic syndromes Engel AG, Curr Opin Pharmacol 2005

115. Myasthenia Gravis Drachman , Trends Neurosci 1983

116. Hereditary hyperekplexia Human startle disease – exaggerated startle response. Normal startle - blinking, grimacing, neck flexion, and arm abduction & flexion. Overreaction to unexpected visual, auditory, or tactile stimuli. Consciousness is preserved during the attacks. May be cause apneic episodes & death. Mutations in GLRA1 and GLRB gene. Bradley’s neurology in clinical practice 7 th edition

117. Mutations in glycine receptor Bradley’s neurology in clinical practice 7 th edition

118. GABA receptors Mutations encoding – α1 subunit of GABA A receptors Autosomal dominant Juvenile myoclonic epilepsy, Sporadic childhood absence seizures Idiopathic generalized epilepsy. Pathophysiology Loss-of function of GABA A receptors Decreased sensitivity to GABA Reduced total cell surface expression Increased retention of the receptor in the endoplasmic reticulum Bradley’s neurology in clinical practice 7 th edition

119. Rasmussen’s encephalitis Etiology: Glutamate receptor autoimmunity. Age of onset: Variable, usually young children Clinical characteristics: Intractable focal epilepsy, often with epilepsia partialis continua (EPC), and progressive hemiparesis followed by progressive intellectual decline. EEG: Slowing ipsilateral to hemiatrophy. EPC may not have EEG correlate. Bradley’s neurology in clinical practice 7 th edition

120. Glial channelopathy Only one glial channelopathy - X-linked Charcot-Marie-Tooth disease. Characterised by a progressive motor and sensory neuropathy. Mutations of the connexin 32 gene. Connexin 32 is expressed in Schwann cells. Due to impaired gap junction function causing disruption of cytoplasmic homeostasis in schwann cells. The lancet, 2002

121. Drugs and channels

122. Drugs and channels

123. Conclusion In all cells ion channels permit the flow of ions across an otherwise impermeable membrane. In nerve and muscle cells ion channels are important for controlling the rapid changes in membrane potential. Channels are also important targets in various diseases. The genetic channelopathies have helped us to understand the pathogenesis of several classes of disease. Many other ion channels are likely to be implicated in neurological diseases in the future. Phenotypic range associated with individual channels is likely to be broader.

124. Thank you